Synthesis and use of anti-reverse mRNA cap analogues

ABSTRACT

The ability to synthesize capped RNA transcripts in vitro has been of considerable value in a variety of applications. However, one-third to one-half of the caps have, until now, been incorporated in the reverse orientation. Such reverse caps impair the translation of in vitro-synthesized mRNAs. Novel cap analogues, such as P 1 -3′-deoxy-7-methylguanosine-5′ P 3 -guanosine-5′ triphosphate and P 1 -3′-O,7-dimethylguanosine-5′ P 3 -guanosine-5′ triphosphate, have been designed that are incapable of being incorporated into RNA in the reverse orientation. Transcripts produced with SP6 polymerase using “anti-reverse” cap analogues were of the predicted length. Analysis of the transcripts indicated that reverse caps were not formed. The in vitro translational efficiency of transcripts with the novel “anti-reverse” cap analogues was significantly higher than that of transcripts formed with conventional caps.

This application is a divisional of patent application Ser. No.10/150,718, filed May 17, 2002, now U.S. Pat. No. 7,074,596, issued Jul.11, 2006; which claimed the benefit of the Mar. 25, 2002 filing date ofprovisional patent application Ser. No. 60/367,404 under 35 U.S.C. §119(e).

The development of this invention was partially funded by the UnitedStates Government under grant number GM20818 awarded by the NationalInstitutes of Health. The United States Government has certain rights inthis invention. The development of this invention was partially fundedby the Government of Poland under grant number 6 P04A 055 17 awarded bythe Polish Committee for Scientific Research (KBN).

In eukaryotes, the 5′ end of most mRNA is blocked, or “capped.” Inaddition, there are some other forms of RNA that are also capped. Thecap contains a 5′-5′ triphosphate linkage between two nucleotides, andalso contains methyl groups. The capping of RNA promotes its normalfunction in cells.

The ability to synthesize capped RNA molecules in vitro is thereforeuseful, because it allows workers to prepare RNA molecules that behaveproperly as mRNA transcripts in a variety of in vitro applications. Suchapplications include both research applications and commercialproduction of certain polypeptides in an in vitro translation system,for example the production of polypeptides containing an “unnatural”amino acid at a specific site.

The method most frequently used to make capped RNAs in vitro is totranscribe a DNA template with either a bacterial RNA polymerase or abacteriophage RNA polymerase in the presence of all four ribonucleosidetriphosphates and a cap dinucleotide such as m⁷G(5′)ppp(5′)G. Thepolymerase initiates transcription with a nucleophilic attack by the3′-OH of the Guo moiety in m⁷GpppG on the α-phosphate of the nexttemplated nucleoside triphosphate, resulting in the initial productm⁷GpppGpN. The alternative, GTP-initiated product pppGpN is suppressedby setting the ratio of m⁷GpppG to GTP between 5 and 10 in thetranscription reaction mixture.

Synthetic RNAs may be synthesized by cell-free transcription of DNAtemplates. See R. Contreras et al., “Simple, efficient in vitrosynthesis of capped RNA useful for direct expression of clonedeukaryotic genes,” Nucl. Acids Res., vol. 10, pp. 6353-6362 (1982); J.Yisraeli et al., “Synthesis of long, capped transcripts in vitro by SP6and T7 RNA polymerases, pp. 42-50 in J. Dahlberg et al. (Eds.), Meth.Enzymol., vol.180., pp. 42-50 (1989); and D. Melton et al., “Efficientin vitro synthesis of biologically active RNA and RNA hybridizationprobes from plasmids containing a bacteriophage SP6 promoter,” Nucl.Acids Res., vol.12, pp. 7035-7056 (1984).

Capped RNAs thus produced are active in in vitro splicing reactions. SeeM. Konarska et al., “Recognition of cap structure in splicing in vitroof mRNA precursors. Cell, vol. 38, pp. 731-736 (1984); and I. Edery etal., “Cap-dependent RNA splicing in a HeLa nuclear extract,” Proc. Natl.Acad. Sci. USA, vol. 82, pp. 7590-7594 (1985).

Capped mRNAs are translated more efficiently than are non-capped mRNAs.See S. Muthukrishnan et al., “5′-Terminal 7-methylguanosine ineukaryotic mRNA is required for translation,” Nature, vol. 255, pp.33-37 (1975); L. Chu et al., “Paradoxical observations on the 5′terminus of ovalbumin messenger ribonucleic acid,” J. Biol. Chem., vol.253, pp. 5228-5231 (1978); E. Darzynkiewicz et al., “β-Globin mRNAscapped with m⁷G, m₂ ^(2.7)G or m₃ ^(2.2.7)G differ in intrinsictranslation efficiency,” Nucl. Acids Res., vol 16, pp. 8953-8962 (1988);and E. Darzynkiewicz et al., “Inhibition of eukaryotic translation bynucleoside 5′-monophosphate analogues of mRNA 5′-cap: Changes in N7substituent affect analogue activity,” Biochem., vol. 28, pp. 4771-4778(1989).

5═-Unmethylated mRNAs have been reported to be translationally lessactive than 5′-methylated mRNAs. See G. Both et al.,“Methylation-dependent translation of viral messenger RNAs in vitro,”Proc. Natl. Acad. Sci. USA, vol. 72, pp. 1189-1193 (1975).

E. Darzynkiewicz et al., “Chemical synthesis and characterization of7-methylguanosine cap analogues,” Biochem., vol. 24, pp.1701-1707 (1985)reported the synthesis of derivatives of 7-methylguanosine 5′-phosphatethat were modified in the ribose moiety by 2′-O or 3′-O-methylation, orby conversion to the 2′-deoxy or arabinosyl form, and reported thatthese derivatives retained cap analogue activity.

F. Sanger et al., “DNA sequencing with chain-terminating inhibitors,”Proc. Natl. Acad. Sci. USA, vol. 74, pp. 5463-5467 (1977) reported amethod for determining DNA nucleotide sequences with 2′,3′-dideoxy andarabinonucleoside analogues of normal deoxynucleoside triphosphates, inwhich the analogs act as specific chain-terminating inhibitors of DNApolymerase.

M. Kadokura et al. 1997, “Efficient synthesis of γ-methyl-cappedguanosine 5′-triphosphate as a 5′-terminal unique structure of U6 RNAvia a new triphosphate bond formation involving activation of methylphosphorimidazolidate using ZnCl₂ as a catalyst in DMF under anhydrousconditions,” Tetrahedron Lett., vol. 38, pp. 8359-8362 (1997) reportedthe synthesis of CH₃pppG from GDP and the imidazolide of methylphosphate in DMF, obtaining a yield of 39% in the absence of ZnCl₂, anda yield of 98% in the presence of ZnCl₂.

A. Pasquinelli et al., “Reverse 5′ caps in RNAs made in vitro by phageRNA polymerases,” RNA, vol. 1, pp. 957-967 (1995) reported thatbacteriophage polymerases also use the 3′-OH of the 7-methylguanosinemoiety of m⁷GpppG to initiate transcription, demonstrating thatapproximately one-third to one-half of RNA products made with this capanalogue actually contain the “reverse cap” Gpppm⁷GpN. Suchreverse-capped RNA molecules behave abnormally. The same authorsreported that when reverse-capped pre-U1 RNA transcripts were injectedinto Xenopus laevis nuclei, they were exported more slowly than naturaltranscripts. Similarly, cytoplasmic reverse-capped U1 RNAs in thecytoplasm were not properly imported into the nucleus. The presence of acap on mRNA strongly stimulates translation of an mRNA transcript intoprotein. To the knowledge of the present inventors, there have been noprevious reports directly addressing whether, and at what rate,reverse-capped mRNAs are translated into protein. However, based on whatis known about recognition of the cap structure by eIF4E, one wouldexpect reverse-capped mRNAs to be translated no more efficiently thanuncapped RNAs.

Z. Peng et al., “Synthesis and application of a chain-terminatingdinucleotide mRNA cap analog,” Org. Lett., vol. 4, pp. 161-164 (2002;published on the Web, December 2001; and including the supportinginformation for this article as reprinted from the journal's web site)reported the synthesis of a chain-terminating mRNA cap dinucleotide,3′-O-Me-m⁷G(5′)pppG, and its use in the in vitro transcription ofhomogeneously capped RNA. Computer modeling was said to indicate thatRNA capped with the compound would be a substrate for cap-dependenttranslation.

Because existing synthetically capped RNAs contain about one-third toone-half reverse caps, the overall translational activity of such a RNApreparation is reduced considerably. Other functional properties of themRNA may also be affected. There is a previously unfilled need for a wayto prepare capped RNA molecules in vitro, in which all or essentiallyall the caps have the proper orientation.

We have discovered and synthesized cap analogues that will not beincorporated into an mRNA molecule in the reverse orientation. Inexperiments in which we synthesized and tested two prototype“anti-reverse” cap analogues (ARCAs), we found that both wereexclusively incorporated into mRNA molecules in the correct orientation.Furthermore, both behaved like natural RNA caps in interactions with thetranslational machinery. The resulting mRNAs were considerably moreactive translationally than are traditional in vitro-prepared RNAscontaining a mixture of caps in both the correct and the reversedorientations.

Transcription by bacteriophage RNA polymerases in the presence ofm⁷GpppG is initiated with a nucleophilic attack by the 3′-OH of eitherthe m⁷Guo moiety or of the Guo moiety on the electrophilic α-phosphateof the first templated nucleoside triphosphate. We eliminated one ofthese two 3′-OH groups, so that the nucleophilic attack would causeincorporation only in the correct orientation. We have made twoprototype ARCAs. In the case ofP¹-3′-deoxy-7-methylguanosine-5′P³-guanosine-5′triphosphate (FIG. 1,Compound 9, henceforth abbreviated m⁷3′dGpppG), we substituted an —H forthe 3′-OH. In the case of P¹-3′-O,7-dimethylguanosine-5′ P³-guanosine-5′triphosphate (FIG. 1, compound 10, henceforth abbreviated m₂^(7,O3′)GpppG), we instead substituted a —OCH₃ for the 3′-OH.

We also developed new coupling strategies to synthesize the prototypeARCAs. To avoid preparing imidazole derivatives from 7-methylatedsubstrates, the activation of which can be difficult, we developed a newcoupling strategy involving guanosine 5′-phosphorimidazolide and themodified 7-methylated nucleoside diphosphate. We obtained high yields byconducting the coupling reaction in the presence of ZnCl₂ instead ofMn²⁺, and by using anhydrous dimethylformamide (DMF) instead of water-asa solvent. See FIG. 1, depicting schematically the synthesis of“anti-reverse” cap analogs. (In FIG. 1, “ImGMP” refers to guanosine5′-imidazolide monophosphate.)

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthesis of “anti-reverse” cap analogs.

FIG. 2 depicts an analysis of in vitro-synthesized RNAs by enzymaticdigestion and anion exchange HPLC.

FIG. 3 depicts the inhibition of translation by ARCAs compared withm⁷GpppG and GpppG.

FIG. 4 depicts the translational activity of ARCA-capped mRNAs comparedto that of other RNAs.

MATERIALS AND METHODS

Synthesis of Mono- and Dinucleotides

EXAMPLE 1

3′-Deoxyguanosine 5-monophosphate (Compound 3). 3′-Deoxyguanosine(Compound 1, commercial product from Sigma, 50 mg, 0.19 mmol) wasstirred overnight with trimethylphosphate (2 mL) and phosphorusoxychloride (53 μL, 0.57 mmol) at 6° C. The reaction was quenched byadding 20 mL of water and neutralizing with 1 M NaHCO₃. DEAE-Sephadexchromatography using a linear gradient of 0-0.9 M TEAB afforded Compound3 (yield: 45 mg, 43%).

EXAMPLE 2

3′-O-Methylguanosine 5′-monophosphate (Compound 4) was obtained by aprocedure analogous to that for Compound 3, but instead starting with 59mg of 3′-O-methylguanosine (Compound 2), which was prepared by themethod of J. Kusmierek et al., “A new route to 2′(3′)-O-alkyl purinenucleosides,” Nucl. Acids Res. Special Publ. No. 4, pp. s73-s77 (1978)(yield: 80 mg, 69%).

EXAMPLE 3

3′-Deoxyguanosine 5′-diphosphate (Compound 5). Compound 3 (55 mg, TEAsalt, 0.1 mmol), imidazole (34 mg, 0.5 mmol) and 2,2′-dithiodipyridine(Aldrich, 44 mg, 0.2 mmol) were mixed in anhydrous DMF (1.2 mL) and TEA(14 μL). Triphenylphosphine (52 mg, 0.2 mmol) was added, and the mixturewas stirred for 5 h at room temperature. The mixture was placed in acentrifuge tube, and sodium perchlorate (49 mg, anhydrous) dissolved inacetone (6 mL) was added. After cooling for 2 h in a refrigerator, themixture was centrifuged and the supernatant was discarded. Theprecipitate was ground with a new portion of acetone, cooled andcentrifuged again. The process was repeated once more, and theprecipitate was dried in a vacuum desiccator over P₄O₁₀. The imidazolidethus obtained was dissolved in 1.2 mL of DMF, and 200 mg oftriethylammonium phosphate was added. (The latter was prepared from TEAand phosphoric acid followed by drying over P₄O₁₀ in a desiccator toobtain a semicrystalline mass.) Finally, 80 mg of ZnCl₂ were added, andthe reaction mixture was stirred at room temperature for 6.5 h, pouredinto a beaker containing a solution of 250 mg EDTA in 15 mL water, andneutralized with 1 M NaHCO₃. Chromatographic isolation on aDEAE-Sephadex column using a linear gradient of 0-1 M TEAB gave Compound5 (yield: 41 mg, 66%).

EXAMPLE 4

3′-O-Methylguanosine 5′-diphosphate (Compound 6) was obtained by aprocedure analogous to that for Compound 5, except starting from 58 mgof Compound 4 (yield: 32 mg, 49%).

EXAMPLE 5

3′-Deoxy-7-methylguanosine 5′-diphosphate (Compound 7). Compound 5 (34mg, 0.055 mmol) was mixed with 1 mL of dimethylsulfoxide, 1 mL of DMF,and 100 μL of methyl iodide at room temperature. After 3 h the reactionmixture was treated with 30 mL of cold water and extracted three timeswith 10-mL portions of diethyl ether. After neutralization with NaHCO₃,chromatographic separation of the aqueous phase on DEAE-Sephadex, usinga linear gradient of 0 to 0.8 M TEAB, gave Compound 7 (yield: 10 mg, 28%).

EXAMPLE 6

3′-O, 7-Dimethylguanosine 5′-diphosphate (Compound 8) was obtained by aprocedure analogous to that for Compound 7, except that the startingmaterial was 66 mg of Compound 6 (yield: 64 mg, 95%).

EXAMPLE 7

P¹-3′-Deoxy-7-methylguanosine-5′ P-guanosine-5′ triphosphate (Compound9). GMP (purchased from Sigma, converted to the TEA salt, 29 mg, 0.05mmol), imidazole (17 mg, 0.25 mmol), and 2,2′-dithiodipyridine (22 mg,0.1 mmol, purchased from Aldrich) were mixed in anhydrous DMF (1.2 mL)and TEA (7 μL). Triphenylphosphine (26 mg, 0.1 mmol) was added, and themixture was stirred for 5 h at room temperature. The mixture was placedin a centrifuge tube, and sodium perchlorate (25 mg, anhydrous)dissolved in acetone (6 mL) was added. The procedure for washing theprecipitate with acetone and drying over P₄O₁₀ was the same as forCompound 5. The imidazolide of GMP thus obtained was dissolved in DMF(1.2 mL), and Compound 7 (10 mg, TEA salt, 0.015 mmol) was added. NextZnCl₂ (40 mg) was added. The mixture was stirred at room temperatureovernight, poured into a beaker containing a solution of 125 mg of EDTAin 15 mL of water, and neutralized with 1 M NaHCO₃. Chromatographicisolation as for Compound 5 gave Compound 9 (13 mg, 88% based on theamount of Compound 7 used).

EXAMPLE 8

P¹-3′-O, 7-Dimethylguanosine-5′P³-guanosine-5′triphosphate (Compound 10)was prepared from GMP and Compound 8 (34 mg) by a procedure analogous tothat for Compound 9 (yield: 23 mg, 78%).

EXAMPLES 9 & 10

The final products (Compounds 9 and 10) were converted to their Na⁺salts by ion exchange on a small column of Dowex 50 W×8 (Na⁺ form),followed by evaporation of the eluates to a small volume, precipitationwith ethanol, and centrifugation to give amorphous white powders.Parameters from the ¹H NMR spectra of Compounds 9 and 10 are shown inTables 1 and 2 below.

EXAMPLE 11

7-Methylguanosine 3′,5′-diphosphate. Guanosine 3′,5′-diphosphate wasmethylated to make the chromatographic standard pm⁷Gp (FIG. 2) by thesame procedure as used for Compound 7.

EXAMPLE 12

Column Chromatography

Both final products (Compounds 9 and 10, FIG. 1) and intermediatenucleotides (Compounds 3-8) were isolated from reaction mixtures bycolumn chromatography on DEAE-Sephadex (A-25, HCO₃ ⁻ form) using alinear gradient of triethylammonium bicarbonate (TEAB), pH 7.5, inwater. Fractions were collected, and products peaks (monitored at 260nm) were pooled and evaporated to dryness, with ethanol added repeatedlyto remove the TEAB buffer. The products were obtained as TEA salts.

The purity of intermediates and products was monitored at 260 nm byanalytical HPLC using a Spectra-Physics SP8800 apparatus on a 25-cmLC-18-T reverse phase column (Supelco). The mobile phase was a lineargradient of methanol from 0 to 25% in 0.1 M KH₂PO₄, pH 6, over 15 minwith flow rate of 1.3 mL/min.

Mono- and dinucleotides obtained by enzymatic digestion of invitro-synthesized RNAs were analyzed by HPLC using a Waters 625instrument with a 996 PDA detector on a 4.5×250 mm Partisil 10SAX/25column (Whatman). The program for elution of nucleotides comprised waterfor the first 5 min; a linear gradient of 87.5 to 500 mM KH₂PO₄, pH 3.5,over 35 min; a linear gradient of 87.5 to 500 mM of KH₂PO₄ over 30 min;and isocratic elution at 500 mM KH₂PO₄ for 21 min—all at a flow rate of1 mL/min.

EXAMPLE 13

Spectroscopy

¹H NMR and ¹³C NMR spectra were recorded on a Varian UNITY plus 500 MHzinstrument in dimethylsulfoxide-d₆ (for nucleoside intermediates) or D₂O(for mono- and dinucleotides). Absorption spectra were obtained on aCary 3E spectrophotometer.

¹H NMR spectra for Compounds 9 and 10 were run at 25° C. at 1.4 mg/0.7mL and 0.4 mg/0.7 mL in D₂O, respectively. Conformations of the sugarmoieties were derived from the vicinal ¹H-¹H coupling constants.Conformations of the phosphate groups were determined from the ¹H-³¹Pcoupling constants.

EXAMPLES 14 & 15

In vitro Synthesis of RNA

Two lengths of RNA, either uncapped or capped with one of the capanalogues, were synthesized by in vitro transcription. The DNA templateused for both lengths of RNA was pSP-luc+ (Promega), which contains anSP6 bacteriophage promoter and a sequence encoding luciferase. Togenerate the short RNAs (43 bases exclusive of the cap), the plasmid wasdigested with Ncol. To generate the long RNAs (1706 bases, containingthe entire luciferase coding region), the plasmid was digested withEcoRI. A typical 20 μL in vitro transcription reaction contained 40 mMTris-HCl, pH 7.9, 6 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 2 μg BSA, 20units of RNasin (Promega), 0.5 mM ATP, 0.5 mM CTP, 0.5 mM UTP, 0.1 mMGTP, 1 mM cap analogue (GpppG, m⁷GpppG, m⁷3′dGpppG, or m₂^(7,O3′)GpppG), 0.2-1.0 μg DNA, and 20 units of SP6 polymerase(Promega). Reactions to synthesize the short RNAs also contained 28 μCiof [α-³²P]ATP (ICN), and those to synthesize the long RNAs contained 0.8μCi of [α-³²P]CTP (ICN). Reaction mixtures were incubated for 60 min at37° C., extracted with phenol and chloroform, and the solution was made2 M in sodium acetate. The nucleic acids were then precipitated with 3volumes of ethanol on dry ice for 5 min, and the mixture was centrifugedat 14,000 rpm for 30 min. The resulting pellet was dissolved in water,and the solution was made 0.2 M in sodium acetate. The nucleic acid wasprecipitated with 2.5 volumes of ethanol at 4° C. for 30 min, and themixture was centrifuged at 14,000 rpm for 30 min. The pellet was allowedto air-dry and then dissolved in diethylpyrocarbonate-treated water.

EXAMPLE 16

Enzymatic Digestion of RNAs

The short RNAs were digested with 67 U RNase T2 (Life Technologies) in15 μL of 0.14 M sodium acetate, pH 4.6, at 37° C. for 60 min. In somecases, the RNAs were subjected to a two-step digestion instead. Thefirst digestion was with 10 U TAP (tobacco acid pyrophosphatase)(Epicentre Technologies) in 5 μL of 50 mM sodium acetate, pH 6.0, 1 mMEDTA, 0.1% β-mercaptoethanol, and 0.01% Triton X-100 at 37° C. for 60min. The digestion was continued for 60 min at 37° C. with 67 U RNase T2in a final volume of 16 μL of 0.12 M sodium acetate, pH 4.6. Sampleswere analyzed without further treatment by anion exchange HPLC asdescribed above.

EXAMPLE 17

Cell-Free Translation

A micrococcal nuclease-treated RRL (rabbit reticulocyte lysate) systemwas used for in vitro translation as described in A. Cai et al.,“Quantitative assessment of mRNA cap analogs as inhibitors of in vitrotranslation,” Biochemistry, vol. 38, pp. 8538-8547 (1999). In somecases, the mRNA used in this system was natural rabbit globin mRNA, andprotein synthesis was measured by incorporation of [³H]Leu into atrichloroacetic acid-precipitable form. In other cases, the mRNA wasluciferase mRNA (the long form), synthesized in vitro as describedabove, and protein synthesis was assayed by measuring luciferaseactivity using beetle luciferin (Promega) as a substrate, and a Monolite2010 luminometer to measure light emission.

The ability of cap analogues to inhibit cell-free translation in the RRLsystem programmed with globin mRNA was measured as described in Cai etal. (1999). Data were fit by least squares minimization to a theoreticalrate equation. The concentrations of cap analogue solutions weremeasured by UV absorption at pH 7.0 using the following parameters for λand ε_(M), respectively: GpppG, 251 nm, 25.5×10³; m⁷GpppG, m⁷3′dGpppG,or m₂ ^(7,O3′)GpppG, 255 nm, 22.6×10³.

Results

¹³C NMR and UV spectra for intermediates were in good agreement with thepredicted structures (data not shown). The ¹H NMR assignments of protonsin both prototype ARCAs confirmed their chemical structures (Table 1).Two sets of sugar ¹H signals in each spectrum pointed to dinucleotides.The presence of methyl signals at 4.068 ppm (Compound 10) and 4.027(Compound 9), together with disappearance of the H(8) resonances due toexchange for solvent deuterium, testified to the presence of7-methylguanine. In the case of Compound 10, the additional methyl groupwas observed at 3.483 ppm, accompanied by a characteristic upfield shiftof the H3′ signal. Lack of the 3′-hydroxyl in Compound 9 gave thecharacteristic “deoxy” pattern of H3′/H3″ at 2.086-2.148 ppm, withfurther scalar couplings to H4′ and H2′. TABLE 1 ¹H NMR chemical shiftsin ppm versus internal sodium 3-trimethylsilyl- [2,2,3,3-D₄]-propionatem⁷3′dGpppG m₂ ^(7,O3′)GpppG (Compound 9) (Compound 10) m⁷3′dG G m₂^(7,O3′)G G H8 —^(a) 8.016 —^(a) 7.990 H1′ 5.796 5.776 5.864 5.785 H2′4.587 4.650 4.682 4.687 H3′ 2.148 4.473 4.109 4.473 H3″ 2.086 — — — H4′4.728 4.346 4.428 4.339 H5′ 4.460 4.26^(b) 4.384 4.278 H5″ 4.1964.26^(b) 4.219 4.239 CH₃ 4.027 — 4.068 (N7) — 3.483 (3′O)^(a)deuterated^(b)signal overlapping

Table 2 provides NMR information concerning conformational parameters.These data reflected populations of the N form in the N⇄S dynamicequilibrium of the sugar ring, populations of the +sc (gauche-gauche)conformer about C4′-C5′, and populations of the ap (gauche′-gauche′)conformer of the phosphate group. The 7-substituted Guo moieties showedthe characteristic preference for the N conformer, up to 100% in thecase of m₂ ^(7,O3′)Guo, as opposed to Guo, in which the S conformerdominates. The preference for +sc was also more pronounced in the7-substituted guanosines. The conformation of the Guo moiety of ARCAswas similar to that of Guo in normal caps, in which about 64% has beenreported to be in the S form (36% N) and about 63% in the +sc form.Thus, m₂ ^(7,O3′)Guo and m⁷3′dG both displayed conformational featuresthat were characteristic of m⁷Guo rather than of Guo. TABLE 2 ¹H-¹H and¹H-³¹P coupling constants in Hz (±0.2), and conformer populations (±5%)in the dynamic equilibria N

S of the sugar ring, and about C4′-C5′ (% +sc) and C5′-O5′ (% ap) bondsm⁷3′dGpppG m₂ ^(7,O3′)GpppG (Compound 9) (Compound 10) m⁷3′dG G m₂^(7,O3′)G G J(1′, 2′)  0.0^(a)  6.2  4.0  6.3 J(2′, 3′)  4.5  5.2  5.0 5.1 J(2′, 3″)  0.0^(a) — — — J(3′, 3″)  14.2 — — — J(3′, 4′)  10.4  3.7 5.1  3.6 J(3″, 4′)  5.1 — — — J(4′, 5′)  3.0^(b)  4.0^(b)  3.0  4.1J(4′, 5″)  2.7  4.0^(b)  2.6  4.2 J(5′, 5″) 11.6 b 11.5 11.8 J(5′, P) 5.0  6.0^(b)  4.4  5.4 J(5″, P)  5.8  6.0^(b)  5.9  6.5 J(4′, P) 1.0^(b)  1.0^(b)  1.0^(b)  1.0^(b) % N 100 37 56 36 % +sc^(c)  8055^(b) 80 54 % ap^(d)  72 66^(b) 74 66^(a)less than the line width, ˜1 Hz^(b)approximate value^(c)+synclinal, i.e., O5′ in gauche orientation to O4′ and C3′^(d)antiperiplanar i.e., P5′ in trans orientation to C4′

EXAMPLE 18

Synthesis of ARCA-Capped RNA Transcripts

We tested the prototype ARCAs in an in vitro transcription system. Atemplate DNA was first generated by digesting the plasmid pSP-luc+ withEcoRI. The theoretical size of an RNA transcript from this templateshould be 1706 bases, which was consistent with the approximate size ofthe products that was observed by electrophoretic migration fromreactions carried out in the presence of [α-³²P]ATP and either GpppG,m⁷GpppG, m⁷3′dGpppG, or m₂ ^(7,O3′)GpppG. (data not shown). Samples wererun on a 1% agarose gel containing 0.12 M formaldehyde in 0.4 M3-(N-morpholino)propanesulfonic acid, pH 7.0, 0.1 M sodium acetate, and0.01 M EDTA at 70 mA for 5 h. A Phosphorimage was obtained with aMolecular Dynamics Storm 860 instrument. Standards used for comparisonwere rabbit 28S rRNA, 18S rRNA, and β-globin mRNA.

In six separate experiments, the yield of RNA product in the presence ofARCAs was not significantly different from the yield in the presence ofm⁷GpppG.

EXAMPLE 19

Analysis of Cap Orientation in ARCA-Capped RNA Transcripts

The structure of the ARCAs was designed to prevent incorporation intoRNA in the reverse orientation. We verified this property experimentallyby digesting RNAs capped with ARCAs with RNase T2 and TAP. To obtain ahigher proportion of radioactivity in the cap versus the internalpositions, a shorter DNA template was produced by cleaving pSP-luc+ withNcol instead of EcoRI. This digestion was expected to yield an RNAproduct of 43 bases (plus the cap). The size of this product wasconfirmed by polyacrylamide gel electrophoresis in Tris/borate/EDTA/urea(data not shown).

RNase T2 digests RNA with no base specificity. Thus, it was expected togenerate primarily 3′-NMPs from this RNA. Those nucleotide residues thatwere located 5′ to an A residue would acquire a ³²P-labeled 3′-phosphateby nearest-neighbor transfer. The pyrophosphate bonds in the cap,however, are not susceptible to RNase T2. Since the first nucleotideresidue after the cap in the synthetic RNA is an A, the α-phosphate of[α-³²P]ATP would be transferred to the cap following RNase T2 digestion.Thus, for RNAs initiated in the normal orientation with m⁷GpppG, theproduct was m⁷GpppGp* (where p* denotes radioactive ³²p). The RNaseT2-digestion products expected from RNAs initiated with GTP or with thefour cap analogues in either normal or reverse orientations are shown inTable 3. TABLE 3 Predicted and observed cap structures from invitro-synthesized mRNAs after enzymatic digestion Possible 5′ endlabeled Product Cap Possible transcription digestion products observeddinucleotide Orientation¹ products RNase T2 RNase T2 + TAP RNase T2 +TAP None N/A pppGP*Ap(Np)₄₀C pppGp* pGp* 100% GpppG N/A GpppGp*Ap(NP)₄₀CGpppGp* pGp* 100% m⁷GpppG Normal m⁷GpppGp*Ap(Np)₄₀C m⁷GpppGp* pGp* 67%Reverse Gpppm⁷Gp*Ap(Np)₄₀C Gpppm⁷Gp* pm⁷Gp* 33% m⁷3′dGpppG Normalm⁷3′dGpppGp*Ap(Np)₄₀C m⁷3′dGpppGp* pGp* 100% ReverseGpppm⁷3′dGp*Ap(Np)₄₀C Gpppm⁷3′dGp* pm⁷3′dGp* 0% m₂ ^(7,O3′)GpppG Normalm₂ ^(7,O3′)GpppGp*Ap(Np)₄₀C m₂ ^(7,O3′)GpppGp* pGp* 100% Reverse Gpppm₂^(7,O3′)GP*Ap(Np)₄₀C Gpppm₂ ^(7,O3′)Gp* pm^(7,O3′)Gp* 0%¹“Normal” orientation means that the 3′-OH of Guo in the structurem⁷G(5′)ppp(5′)G (or its analogues) is attached to the first nucleotideresidue in the RNA chain by a 3′-5′phosphodiester linkage. “Reverse”orientation means that the 3′-OH of m⁷Guo is the point of attachment.²Radioactive atoms (³²P) are indicated by *.

The RNase T2-digestion products of normal and reverse m⁷GpppG-cappedRNAs (m⁷GpppGp* and Gpppm⁷Gp*, respectively) have identical masses andcharges; they would therefore be expected to elute from an anionexchange column at nearly the same time. However, TAP digestion ofnormal and reverse-capped mRNAs should yield two alternate labeledproducts, pGp* and pm⁷Gp*, that differ in both charge and mass, becausethe m⁷ group confers a positive charge on G. The nucleotides pm⁷3′dGp*and pm₂ ^(7,O3)′Gp* have the same charge as pm⁷Gp*. Thus, although RNaseT2 digestion alone would not be expected to distinguish between normaland reverse orientations, the combination of RNase T2 and TAP should doso (see Table 3).

RNA was synthesized from the short DNA template in the presence of: (1)[α-³²P]ATP; (2) the other three NTPs (nonradioactive); and (3) eitherGpppG, m⁷GpppG, m⁷3′dGpppG, m₂ ^(7,O3′)GpppG, or no cap analogue. Theproducts were digested with RNase T2 and resolved by anion exchangeHPLC. FIG. 2 depicts an analysis of the in vitro-synthesized RNAs byenzymatic digestion and anion exchange HPLC. The mRNAs were generated bytranscription of Ncol-digested pSP-luc+ with [α-³²P]ATP and either nocap dinucleotide (panels A, B), GpppG (panels C, D), m⁷GpppG (panels E,F), m⁷3′dGpppG (panels G, H), or m₂ ^(7,O3′)GpppG (panels I, J).Aliquots of 5 to 13 ng of RNA were digested with RNase T2 (left panels),or with both RNase T2 and TAP (right panels). Nucleotides and caps wereseparated on a Partisil 10SAX/25 column developed with a gradient ofpotassium phosphate, pH 3.5. Fractions of 1 mL were collected, and theirCerenkov radiation was determined with a Beckman LS 6500 scintillationcounter. The elution times of the following standard compounds, detectedby UV absorption, are also shown: 3′-CMP; 3′-UMP; 3′-AMP; 3′-GMP;5′-GDP; 5′-GTP; 3′,5′-GDP (pGp); 3′,5′-m⁷GMP (pm⁷Gp); and GpppG.

Uncapped RNA yielded primarily 3′-NMPs (Panel A, 20-30 min) with a smallamount of material that may have been the partially degraded productppGp* (Panel A, 76 min). The expected product pppGp* was not observed.Due to its high negative charge, that species may not have eluted fromthe column. Its presence, however, is likely since RNase T2 plus TAPdigestion yielded pGp* (Panel B, 56 min) where none had existedpreviously (compare Panel A).

In the case of GpppG-capped RNAs, RNase T2 alone yielded a structureeluting at 89 min (FIG. 2, Panel C), likely GpppGp* (the presence of asecond phosphate ester reduces the charge relative to pppGp*). The minorpeak at 77 min may have been the partially degraded product ppGp*.Consistent with these assignments, both compounds disappeared followingTAP digestion, coinciding with the appearance of a new peakcorresponding to pGp* at 56 min (FIG. 2, Panel D). As expected, nopm⁷Gp* (42 min) was formed.

The major, highly-charged RNase T2-resistant product from m⁷GpppG-cappedRNA eluted at 73 min (FIG. 2, Panel E), likely m7GpppGp*. This compoundeluted earlier than the peak at 89 min in Panel C, tentatively assignedthe structure GpppGp*, because of the additional positive charge. Theminor peak at 77 min may be the reverse cap Gpppm⁷Gp*, suggesting thatthe proximity of the 3′-P to the positive charge of the m⁷G ring mayinfluence the charge on the P. These assignments are strengthened by thefact that TAP digestion converted these products to two labeledcompounds that eluted earlier, pGp* (56 min) and pm⁷Gp* (42 min) (FIG.2, Panel F). The ratio of pGp* to pm⁷Gp* suggest that they were derivedfrom the 73- and 77-min peaks of Panel E, respectively.

With the ARCA m⁷3′dGpppG, an RNase T2-resistant product was observed at78 min, likely m⁷3′dGpppGp* (FIG. 2, Panel G). It eluted at nearly thesame time as the compound thought to be m⁷GpppGp* (78 min versus 77 minfor Panels G and E, respectively). Note that where there were two peaksin this region for RNA synthesized with m⁷GpppG (Panel E), there wasonly one peak for RNA synthesized with the ARCA (Panel G), consistentwith the inability of the ARCA to be incorporated in the reverseorientation. After digestion with TAP, the peak at 78 min disappearedand a new one appeared at the elution time of pGp* (Panel H, 56 min).The fact that no pm⁷Gp* appeared at 42 min with the ARCA (Panel H),while it did with m⁷GpppG (Panel F), is further proof that the ARCA wasincorporated in only a single orientation.

The products observed upon digestion of RNA synthesized with the secondARCA, m₂ ^(7,O3′)GpppG (FIG. 2, Panels I and J), eluted almost the sameas those that had been obtained with the m⁷3′dGpppG-capped RNA—again,consistent with the expectation that the ARCA should be incorporated inonly one orientation.

EXAMPLE 20

Competitive Inhibition of Protein Synthesis by ARCAs

One measure of the interaction between a cap analogue and thetranslational machinery is competitive inhibition of protein synthesis.The binding of cap analogues to eIF4E, measured in vitro with purifiedcomponents, and the resulting competitive inhibition of proteinsynthesis in a cell-free translation system have been correlated forseveral different cap analogue structures. We separately tested GpppG,m⁷GpppG, and the two ARCAs for their ability to compete with naturalglobin mRNA for recognition by the translational machinery, and therebyto inhibit translation in an RRL system. GpppG did not act as aninhibitor, and in fact slightly stimulated protein synthesis at lowconcentrations. The two ARCAs, on the other hand, were equally asinhibitory as m⁷GpppG. FIG. 3 depicts the inhibition of translation byARCAs compared with m⁷GpppG and GpppG. Natural rabbit globin mRNA wastranslated at 5 μ/mL in the RRL system, and globin synthesis wasdetected by incorporation of [³H]Leu into protein. The following capanalogues were included during translation at the indicatedconcentrations: GpppG, circles; m⁷GpppG, squares; m⁷3′dGpppG, triangles;and m₂ ^(7,O3′)GpppG, diamonds.

One may compare cap analogues as inhibitors quantitatively, by fitting atheoretical curve to observed translation data. The value of thedissociation constant, K_(I), for the cap analogue·eIF4E complex wasvaried to obtain the best least-squares fit. FIG. 3 depicts such curvesfor m⁷GpppG, m⁷3′dGpppG, and m₂ ^(7,O3′)GpppG, with corresponding K_(I)values of 27.8±12.6, 27.8±7.1, and 14.3±1.9 μM, respectively. Althoughit appeared in this experiment that the m₂ ^(7,O3′)GpppG compound wasmore inhibitory, in a replicate of this experiment the K_(i) values forthe ARCAs did not differ statistically from those of m⁷GpppG.

EXAMPLE 21

Translation of ARCA-Capped mRNAs

Because one-third to one-half of m⁷GpppG was incorporated into RNA inthe reverse orientation, because the novel ARCAs were incorporatedexclusively in the normal orientation, and because the ARCAs wererecognized to the same extent as m⁷GpppG in the translational inhibitionexperiment, we predicted that a homogeneous population of invitro-synthesized ARCA-capped mRNAs should be more activetranslationally than m⁷GpppG-capped mRNAs. We tested this predictionusing luciferase mRNAs that were either uncapped, capped with GpppG,capped with m⁷GpppG, or capped with one of the two ARCAs.

FIG. 4 depicts the translational activity of the ARCA-capped mRNAs.Luciferase mRNAs were synthesized in vitro using SP6 RNA polymerase inthe presence of all four NTPs, and either no cap analogue (circles),GpppG (squares), m⁷GpppG (diamonds), m₂ ^(7,O3′)GpppG (invertedtriangles) or m⁷3′dGpppG (triangles). The RNAs were translated for 60min in the RRL system, and luciferase activity was measured intriplicate by luminometry (RLU, relative light units). Translationalefficiency for each mRNA was estimated from the slopes of the curves ofluciferase activity versus mRNA concentration.

The fact that all the m⁷G-containing mRNAs were translated moreefficiently than the uncapped or GpppG-capped mRNAs indicated that theRRL system we used was highly dependent on the presence of a cap on themRNA. As shown in FIG. 4, we found that the m₂ ^(7,O3′)GpppG-andm⁷3′dGpppGp-capped mRNAs were more efficient in translation thanm⁷GpppG-capped mRNA. In six experiments employing four separate batchesof in vitro-synthesized mRNAs, the mRNAs produced with the novel ARCAswere consistently more active than those produced with m⁷GpppG.

Pasquinelli et al. (1995) found that the extent of reverse cappingvaried between 28% and 48%, depending on the pH of the invitro-transcription reaction. In the experiments whose results are shownin FIG. 2 and summarized in Table 3, the extent of reverse capping wasapproximately 33%. Assuming that the novel ARCAs and normal capsstimulate translation to the same degree, an assumption that seemslikely based on the inhibition data (FIG. 3), we predicted that the(homogeneous) preparation of ARCA-capped mRNA should be more active thanthe (heterogeneous) preparation of m⁷GpppG-capped mRNAs, a predictionthat was consistent with our experimental observations.

These results showed that the novel ARCAs behaved very similarly tonormal cap analogues, except that they were not incorporated into RNAsin the reverse orientation, and that they can cause substantially highertranslational activity. The modifications at the 3′-O-position of m⁷Guodid not appear to substantially alter conformation (Table 2) orinteraction with translational machinery (FIGS. 3 and 4). The ARCAs havethe advantage of being incorporated into RNA exclusively in the normalorientation, but have no apparent disadvantages. To our knowledge, thedegree to which m⁷G is incorporated in place of G in internal positionsof a synthetic RNA chain by bacteriophage polymerases has not beenrigorously determined. Regardless of the level of such misincorporation,the ARCAs should be essentially incapable of donating m⁷G eitherinternally or at the 5′-end. A different type of ARCA, e.g., m₄^(2,2,7,O3′)GpppG or m₃ ^(2,2,7)3′dGpppG, would be useful for in vitrosynthesis of U-type snRNAs with 100% normal cap orientation.

EXAMPLE 22

An Arabinose-Derived ARCA

Anti-reverse mRNA cap analogues may also be derived from arabinose, forexample:

For example, where n=1, this ARCA may be synthesized starting with9-β-D-arabinofuranosylguanine, which is commercially available or whichmay be prepared by the method of Ikehara et al., “Studies of nucleosidesand nucleotides. XLVIII. Purine cyclonucleosides. 29. A new method forthe synthesis of 9-β-arabinofuranosylguanine (Ara G),” J. Carbohydr.Nucleosides Nucleotides, vol. 3, pp. 149-159 (1976), in place of the3′-deoxyguanosine in Example 1.

Although the examples described above employed particular cap analogs,other analogs will also work in practicing the invention, for example:

 1. m₂ ^(7,3′O)GpppG: X = OH, Y = OCH₃, n = 1;  2. m⁷3′dGpppG: X = OH, Y= H, n = 1;  3. m₂ ^(7,2′O)GpppG: X = OCH₃, Y = OH, n = 1;  4.m⁷2′dGpppG: X = H, Y = OH, n = 1;  5. m⁷2′,3′didGpppG: X = H, Y = H, n =1;  6. m₃ ^(7,2′O,3′O)GpppG: X = OCH₃, Y = OCH₃, n = 1;  7.m⁷et^(3′O)GpppG: X = OH, Y = OC₂H₅, n = 1;  8. m⁷et^(2′O)GpppG: X =OC₂H₅, Y = OH, n = 1;  9. m₂ ^(7,3′O)GppppG: X = OH, Y = OCH₃, n = 2;10. m⁷3′dGppppG: X = OH, Y = H, n = 2; 11. m₂ ^(7,2′O)GppppG: X = OCH₃,Y = OH, n = 2; 12. m⁷2′dGppppG: X = H, Y = OH, n = 2; 13.m⁷2′,3′didGppppG: X = H, Y = H, n = 2; 14. m₃ ^(7,2′O,3′O)GppppG: X =OCH₃, Y = OCH₃, n = 2; 15. m⁷et^(3′O)GppppG: X = OH, Y = OC₂H₅, n = 2;16. m⁷et^(2′O)GppppG: X = OC₂H₅, Y = OH, n = 2.

Note that both 2′ and 3′ modifications may be used. These compounds may,for example, be synthesized in a manner generally analogous to thesyntheses described above. For example, the synthesis of alternative 3in the above list (X═OCH₃, Y═OH, n=1) may be conducted in a mannersimilar to that described in the above Examples, starting by replacingthe 3′-O-methylguanosine with 2′-O-methylguanosine in Example 2, thesynthesis of the latter of which is also described in Kusmierek et al.(1978).

Likewise, the synthesis of alternative 4 in the above list (X═H, Y═OH,n=1) may be conducted in a manner similar to that described in the aboveExamples, starting with 2′-deoxyguanosine 5′-diphosphate, which iscommercially available, in Example 3 in lieu of 2′-deoxyguanosine5′-diphosphate.

The synthesis of alternative 5 may be conducted in a similar manner,starting with 2′,3′-dideoxyguanosine (which is commercially available)in lieu of 3′-deoxyguanosine in Example 1.

The synthesis of alternative 6 may be conducted by modifying theprocedure of Kusmierek et al. (1978) by using a large excess ofmethylation reagent to prepare the starting material to use as otherwisedescribed in the Examples, starting with Example 2.

The synthesis of alternative 7 may be conducted by modifying theprocedure of Kusmierek et al. (1978) by using diazoethane (instead ofdiazomethane) as alkylating reagent to prepare the starting material touse as otherwise described in the Examples, starting with Example 2.

The synthesis of alternative 8 may be conducted by modifying theprocedure of Kusmierek et al. (1978) by using diazoethane (instead ofdiazomethane) as alkylating reagent to prepare the starting material touse as otherwise described in the Examples, starting with Example 2.

The synthesis of alternatives 9-16 (n=2) may be conducted as otherwisedescribed in the above Examples, or in the above syntheses ofalternatives 3-8 (n=1), as appropriate, but using GDP instead of GMP inthe steps as otherwise described in Example 7. Likewise, analogues withhighervalues of n may be prepared using guanosine triphosphate,guanosine tetraphosphate, guanosine pentaphosphate, etc. in lieu of GMPin the steps otherwise described in Example 7.

We expect that 2′,3′-dideoxy- and 2′,3′-dimethyl cap analogs willfunction in the present invention. We also expect that introducingadditional phosphate groups into the phosphate bridge (creating, e.g.,dinucleotide tetraphosphates or even penta-, hexa-, or heptaphosphates(n=3, 4, or 5 in the above structure))—will produce compounds that maybe more effective than the triphosphates. Other possible substituents Xand Y include OCH₂CH₃. If Y is OH, then it is preferred that X isneither H nor OH.

The “non-methylated” guanosine in the ARCA may be replaced with anothernucleoside, e.g., uridine, adenosine, or cytosine:

wherein the moiety B is selected from the group consisting of

The synthesis of the ARCAs with these alternative nucleosides may beconducted as otherwise described in the above Examples, or in the abovesyntheses of alternatives 1-16, as appropriate, but using AMP, ADP, ATP,UMP, UDP, UTP, CMP, CDP, CTP, etc. in lieu of GMP in the steps otherwisedescribed in Example 7.

Another alternative is to replace the 7-methyl group with anothersubstituent, such as C₁ to C₄ substituted or unsubstituted alkyl, C₆ toC₈ substituted or unsubstituted aryl, or C₁ to C₄ substituted orunsubstituted alkoxy, as illustrated in the examples below:

[a] et⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = C₂H₅ n = 1; [b] et⁷3′dGpppG:X = OH, Y = H, Z = C₂H₅ n = 1; [c] bn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z= CH₂C₆H₅ n = 1; [d] bn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₅ n = 1; [e]et⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = C₂H₅ n = 2; [f] et⁷3′dGpppG: X =OH, Y = H, Z = C₂H₅ n = 2; [g] bn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z =CH₂C₆H₅ n = 2; [h] bn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₅ n = 2; [i]pFbn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = CH₂C₆H₄pF n = 1; [j]pFbn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₄pF n = 1; [k]pClbn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = CH₂C₆H₄pCl n = 1; [l]pClbn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₄pCl n = 1; [m]pFbn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = CH₂C₆H₄pF n = 2; [n]pFbn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₄pF n = 2; [o]pClbn⁷m^(3′O)GpppG: X = OH, Y = OCH₃, Z = CH₂C₆H₄pCl n = 2; [p]pClbn⁷3′dGpppG: X = OH, Y = H, Z = CH₂C₆H₄pCl n = 2;

The synthesis of alternatives [a] and [b] above may be carried out, forexample, as otherwise described in the Examples above, starting withExample 5 or 6, and replacing the 100 μL of methyl iodide with 100 μL ofethyl iodide.

The synthesis of alternatives [c] and [d] above may be carried out, forexample, as otherwise described in the Examples above, starting withExample 5 or 6, and replacing the 100 μL of methyl iodide with 100 μL ofbenzyl bromide. See generally M. Jankowska et al., “Synthesis andproperties of new NH₂ and N7 substituted GMP and GTP 5′-mRNA capanalogues,” Collect. Czech. Chem. Commun., vol. 58, pp. S138-S141(1993).

The synthesis of alternatives [e] through [h] above may be carried out,for example, as otherwise described in the Examples above, starting withExamples 5 through 8, and replacing the 100 μL of methyl iodide with 100μL of ethyl iodide or benzyl bromide, as appropriate, and replacing GMPwith GDP.

The synthesis of alternatives [i] through [p] above may be carried out,for example, as otherwise described in the Examples above, starting withExamples 5 through 8, and replacing the 100 μL of methyl iodide with 100μL of p-chlorobenzyl chloride or p-fluorobenzyl chloride, asappropriate, and replacing GMP with GDP when appropriate. See Jankowskaet al. (1993).

Another possible modification is a methyl or other substitution at theN² position:

where, R may, for example, be H, CH₃, CH₂C₆H₅, CH₂COC₆H₅, CH₂CH₂CH₂OH,CH₂CH═CH₂, or another substituent, such as C₁ to C₄ substituted orunsubstituted alkyl, or C₆ to C₈ substituted or unsubstituted aryl. Suchmodifications may, for example, be made at the beginning of thesynthetic route, in the initial synthesis of the nucleoside, prior tocarrying out the other steps of the synthesis. For example,N²,3′-O-dimethylguanosine 5′-monophosphate may be obtained by aprocedure analogous to that for Compound 3, but instead starting withN²,3′-O-dimethylguanosine, which may be prepared by introduction at thebeginning of methyl groups into the N² position of guanosine by themethod of J. Boryski et al., Nucleosides Nucleotides, vol. 4, pp. 595 ff(1985); or Sekine et al., “A convenient method for the synthesis ofN²,N²-dimethylguanosine by reductive C—S bond cleavage with tributyltinhydride,” J. Org. Chem., vol. 56, pp. 1224-1227 (1991). See also J.Boryski, “Application of the 1,N-2-isopropenoguanosine system forsynthesis of novel N-2-substituted derivatives of guanosine andacyclovir,” Coll. Czech. Chem. Commun., vol. 55 (special issue), pp.85-88 (1990).

Still more generally, compounds in accordance with the present inventionwill include structures such as the following:

wherein the substituents R, X, Y, and Z are as previously described, andthe moiety B is selected from the group consisting of

Miscellaneous

The complete disclosures of all references cited in this specificationare hereby incorporated by reference. Also incorporated by reference arethe complete disclosures of the following publications of the inventors'own work, which are not prior art to the present application: J.Stepinski et al., “Synthesis and properties of mRNAs containing thenovel ‘anti-reverse’ cap analogs 7-methyl(3′-O-methyl)GpppG and7-methyl(3′-deoxy)GpppG,” RNA, vol. 7, pp. 1486-1495 (2001); E.Darzynkiewicz et al., “New ‘anti-reverse’ 5′-mRNA dinucleotide capanalogues (ARCA),” Abstract POTH-035, 27th Meeting of the Federation ofEuropean Biochemical Societies (Lisbon, Portugal, Jun. 30-Jul. 5, 2001);J. Stepinski et al., “Synthesis and properties of ‘anti-reverse’ capanalogues,” Abstract, 6th Meeting of the RNA Society (Banff, Canada, May29-Jun. 3, 2001); and J. Stepinski et al., “Preparation and propertiesof mRNAs capped with the novel ‘anti-reverse’ dinucleotide capanalogues,” Abstract P-13, 4th West Coast Meeting on mRNA Stability andTranslation (Seattle, Wash., Oct. 14-16, 2001). In the event of anotherwise irreconcilable conflict, however, the present specificationshall control.

1. A composition comprising

wherein: X is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; Y is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; n is 1,2,3,4, or5; wherein, if Y is OH and n is 1, then X isneither H nor OH; and B is selected from the group consisting of


2. A composition as recited in claim 1, wherein X is OH, Y is H, and nis
 1. 3. A composition as recited in claim 1, wherein X is OH, Y isOCH₃, and n is
 1. 4. An RNA molecule whose 5′ end incorporates acomposition as recited in claim
 1. 5. An RNA molecule whose 5′ endincorporates a composition as recited in claim
 2. 6. An RNA moleculewhose 5′ end incorporates a composition as recited in claim
 3. 7. Amethod for synthesizing an RNA molecule as recited in claim 4 in vitro;said method comprising reacting ATP, CTP, UTP, GTP, a composition asrecited, and a polynucleotide template; in the presence an RNApolymerase; under conditions conducive to transcription by the RNApolymerase of the polynucleotide template into an RNA copy; whereby someof the RNA copies will incorporate the composition as recited to make anRNA molecule as recited.
 8. A method for synthesizing an RNA molecule asrecited in claim 5 in vitro; said method comprising reacting ATP, CTP,UTP, GTP, a composition as recited, and a polynucleotide template; inthe presence an RNA polymerase; under conditions conducive totranscription by the RNA polymerase of the polynucleotide template intoan RNA copy; whereby some of the RNA copies will incorporate thecomposition as recited to make an RNA molecule as recited.
 9. A methodfor synthesizing an RNA molecule as recited in claim 6 in vitro; saidmethod comprising reacting ATP, CTP, UTP, GTP, a composition as recited,and a polynucleotide template; in the presence an RNA polymerase; underconditions conducive to transcription by the RNA polymerase of thepolynucleotide template into an RNA copy; whereby some of the RNA copieswill incorporate the composition as recited to make an RNA molecule asrecited.
 10. A method for synthesizing a protein or a peptide in vitro,said method comprising translating an RNA molecule as recited in claim 4in a cell-free protein synthesis system, wherein the RNA moleculecomprises an open reading frame, under conditions conducive totranslating the open reading frame of the RNA molecule into the proteinor peptide encoded by the open reading frame.
 11. A method forsynthesizing a protein or a peptide in vitro, said method comprisingtranslating an RNA molecule as recited in claim 5 in a cell-free proteinsynthesis system, wherein the RNA molecule comprises an open readingframe, under conditions conducive to translating the open reading frameof the RNA molecule into the protein or peptide encoded by the openreading frame.
 12. A method for synthesizing a protein or a peptide invitro, said method comprising translating an RNA molecule as recited inclaim 6 in a cell-free protein synthesis system, wherein the RNAmolecule comprises an open reading frame, under conditions conducive totranslating the open reading frame of the RNA molecule into the proteinor peptide encoded by the open reading frame.
 13. A compositioncomprising

wherein n is 1, 2, 3, 4, or
 5. 14. An RNA molecule whose 5′ endincorporates a composition as recited in claim
 13. 15. A method forsynthesizing an RNA molecule as recited in claim 14 in vitro; saidmethod comprising reacting ATP, CTP, UTP, GTP, a composition as recited,and a polynucleotide template; in the presence an RNA polymerase; underconditions conducive to transcription by the RNA polymerase of thepolynucleotide template into an RNA copy; whereby some of the RNA copieswill incorporate the composition as recited to make an RNA molecule asrecited.
 16. A method for synthesizing a protein or a peptide in vitro,said method comprising translating an RNA molecule as recited in claim14 in a cell-free protein synthesis system, wherein the RNA moleculecomprises an open reading frame, under conditions conducive totranslating the open reading frame of the RNA molecule into the proteinor peptide encoded by the open reading frame.
 17. A compositioncomprising

wherein: X is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; Y is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; n is 1, 2, 3, 4, or 5; and Z is selected from the groupconsisting of C₁ to C₄ substituted or unsubstituted alkyl, C₆ to C₈substituted or unsubstituted aryl, or C₁ to C₄ substituted orunsubstituted alkoxy; and wherein, if Z is CH₃ and Y is OH and n is 1,then X is neither H nor OH.
 18. An RNA molecule whose 5′ endincorporates a composition as recited in claim
 17. 19. A method forsynthesizing an RNA molecule as recited in claim 18 in vitro; saidmethod comprising reacting ATP, CTP, UTP, GTP, a composition as recited,and a polynucleotide template; in the presence an RNA polymerase; underconditions-conducive to transcription by the RNA polymerase of thepolynucleotide template into an RNA copy; whereby some of the RNA copieswill incorporate the composition as recited to make an RNA molecule asrecited.
 20. A method for synthesizing a protein or a peptide in vitro,said method comprising translating an RNA molecule as recited in claim18 in a cell-free protein synthesis system, wherein the RNA moleculecomprises an open reading frame, under conditions conducive totranslating the open reading frame of the RNA molecule into the proteinor peptide encoded by the open reading frame.
 21. A compositioncomprising

wherein: X is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; Y is selected from the group consisting of H, OH, OCH₃, andOCH₂CH₃; R is selected from the group consisting of H, CH₃, CH₂C₆H₅,CH₂COC₆H₅, CH₂CH₂CH₂OH, CH₂CH═CH₂, C₁ to C₄ substituted or unsubstitutedalkyl, and C₆ to C₈ substituted or unsubstituted aryl; n is 1, 2, 3, 4,or 5; Z is selected from the group consisting of C₁ to C₄ substituted orunsubstituted alkyl, C₆ to C₈ substituted or unsubstituted aryl, or C₁to C₄ substituted or unsubstituted alkoxy; and B is selected from thegroup consisting of guanine, adenine, uridine, and cytosine; andwherein, if Z is CH₃, and R is H, and Y is OH, and n is 1, and B isguanine; then X is neither H nor OH.
 22. An RNA molecule whose 5′ endincorporates a composition as recited in claim
 21. 23. A method forsynthesizing an RNA molecule as recited in claim 22 in vitro; saidmethod comprising reacting ATP, CTP, UTP, GTP, a composition as recited,and a polynucleotide template; in the presence an RNA polymerase; underconditions conducive to transcription by the RNA polymerase of thepolynucleotide template into an RNA copy; whereby some of the RNA copieswill incorporate the composition as recited to make an RNA molecule asrecited.
 24. A method for synthesizing a protein or a peptide in vitro,said method comprising translating an RNA molecule as recited in claim22 in a cell-free protein synthesis system, wherein the RNA moleculecomprises an open reading frame, under conditions conducive totranslating the open reading frame of the RNA molecule into the proteinor peptide encoded by the open reading frame.